Alternative splicing massively expands the complexity of the human transcriptome and nearly all human genes produce multiple mRNAs that encode distinct proteins. Regulation of splicing is primarily mediated by RNA binding proteins (RBPs) that bind to exonic and intronic sequences and function as splicing enhancers or silencers of nearby splice sites or exons. The net activities of a number of RBPs that bind within or near regulated exons combinatorially determine the splicing outcome. Studies of several well characterized splicing factors have revealed RNA maps of binding that define whether they promote or repress exon splicing. The RNA maps of a collection of splicing factors expressed in a given tissue or cell are therefore predicted to determine global splicing patterns. Such maps will thus define a splicing code of diverse sequence motifs and features that can confidently predict tissue-specific differences in splicing. Recent progress in this area has shown that tissue specific splicing can be predicted on the basis of RNA sequence features and revealed a vast collection of RNA sequence motifs and features that comprise this code. However, many gaps in our understanding of the complete splicing code remain. First, only a minority of the total set of splicing factors are defined. Second, the protein regulators that bindto the most of the sequence motifs that regulate splicing are unknown. Third, a more complete splicing code that can also predict more complex differences in splicing in all cell types and conditions requires substantial new inputs. A first step towards resolving these issues will be to complete the full inventory of all human proteins that directly regulate splicing and determine their cognate binding sequence motifs. We will undertake this step through the following specific aims: 1) Perform high throughput screening (HTS) splicing assays using a comprehensive library of human RNA binding proteins. We will use RNA tethering assays in conjunction with previously validated luciferase-based splicing reporters to screen a library of nearly all human RBPs for the ability to enhance or silence exon splicing from different intronic positions. This screening pipeline and subsequent validation steps will vastly expand the set of known alternative splicing regulators in the human genome. 2) Determination of RNA binding specificities for novel splicing regulators by multiplexed RNA SELEX-Seq. Using an innovative new high throughout method the high affinity binding motifs for all known and novel splicing regulators will be determined using a cell-based method to that couples systematic evolution of ligands by exponential enrichment (SELEX) with high throughput sequencing (RNA-Seq). The results from these studies will provide a comprehensive view of the cis- and trans-regulators of alternative splicing that define the splicing code. When couples with existing experimental and bioinformatics databases will substantially improve our definition of the splicing code and provide new insights into the molecular mechanisms that lead to tissue-specific splicing. PUBLIC HEALTH RELEVANCE: Alternative splicing allows the same gene transcript to be processed into proteins with different functions in different cell types, at different stages of development, and in numerous diseases including cancer. To understand how this process is regulated, we propose to carry out a systematic analysis to define the majority of the proteins that regulate this process and the RNA sequences that they bind. Identification of the complete inventory of protein-RNA regulatory interactions that control alternative splicing on a genome-wide scale will provide fundamental insights with broad applications to our understanding of human health and diseases.